Streptococcus agalactiae strain was isolated from patients with RVVC and named Saga-Eh1. C. albicans strain SC5314, C. albicans SC5314-GFP and S. agalactiae strain Saga-Eh1 were stored in our laboratory. C. albicans strain was cultured on Sabouraud dextrose chloramphenicol agar plate and then maintained in yeast extract peptone dextrose (YPD) medium at 30°C. Saga-Eh1 was cultured on blood plate or Tryptone soya broth (TSB) medium at 37°C (Table 1).

Clinically isolated S. agalactiae strains were identified using VITEK2 COMPACT (BIOMERIEUX), a single colony selected for future identification. PCR amplicons of S. agalactiae 16S ribosomal DNA (16SrDNA) (Forward primer:AGTTTGATCCTGGCTCAG; Revese primer:GGTTACCTTGTTACGACTT) identification were obtained from chromosomal DNA. The sequences of the PCR products were compared with the existing sequences available at https://blast.ncbi.nlm.nih.gov. To further confirmation isolated clinical S. agalactiae strains CAMP detection were positive (Table 1).

Clinically isolated C. albicans strains were identified using VITEK2 COMPACT (BIOMERIEUX), and a single colony selected for future identification. PCR amplicons of C. albicans the internal transcribed spacer (ITS) region (ITS1-ITS4) (ITS1primer:TCCGTAGGTGAACCTGCGG; ITS4 primer:TCCTCCGCTTATTGATATG) were obtained from chromosomal DNA. The sequences of the PCR products were compared with the existing sequences available at https://blast.ncbi.nlm.nih.gov (Table 1).

Hyphal induction was manipulated using a standard method. Strains were grown overnight in liquid YPD at 30°C, and diluted 1:250 in Spider media, YPD+10% fetal bovine serum (FBS), SD+10% FBS and Roswell Park Memorial Institute (RPMI) 1640. Cells were incubated at 37°C and harvested, and hyphal morphology were examined under a microscope at the indicated time points.

Candida albicans cells were added to the upper part of the Transwell chamber (3 μm pore, Costar), and S. agalactiae cells were added to the lower chamber, which allowed us to observe hyphal development of C. albicans at 37°C toward active molecule-containing media or cell-conditioned media at different time points.

Candida albicans cells were harvested using centrifugation at 4000 rpm and 4°C, and cell pellets were resuspended in 200 μL ice-cold extraction buffer [0.1 M LiCl, 0.1 M Tris-HCl (pH 7.5), 5% SDS, 2% β-mercaptoethanol]. The mixtures were transferred to a new microcentrifuge tube that contained 0.3 g of acid-washed glass beads and PCIA (Phenol-Chloroform-isoamyl alcohol) pH 4.5, vortexed at full speed for 5 min and placed immediately on ice for precipitation. Nucleic acid pellets were collected by centrifugation, washed with 70% ethanol, air-dried, and then dissolved in 100 μL nuclease-free water. DNA contaminants were removed with RNase-free DNase I (Promega). The 20-μL reaction mixture included 2^∗SYBR Green Master Mix (High ROX premixed), 5 μL of first-strand cDNA reaction mixture, and 0.4 μM primers. The amplification program and analysis were performed according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green Master Mix (High ROX Premixed) (Vazyme, Nanjing, China) using the primers CBO284 and CBO285 for TEC1, CBO282 and CBO283 for EFG1, CBO280 and CBO281 for BRG1, CBO278 and CBO279 for BCR1, CBO286 and CBO287 for NDT80, CBO288 and CBO289 for ROB1, CBO292, and CBO293 for HWP1 (Table 2).

All animal experiments were approved by Institut Pasteur of Shanghai Chinese Academy of Sciences (IPS) IACUC [A20150013]. All animal experiments were carried out in strict accordance with the regulations in the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China. Animals were euthanized by carbon dioxide inhalation.

Nine to ten-week-old female C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. The mouse model of vaginal infection was established as described elsewhere (Enjalbert et al., 2009; Pietrella et al., 2011). Three days prior to infection a pseudoestrus condition was induced in the mice by administration of subcutaneous injection of 0.2 mg estradiol valerate in 100 μL of sesame oil (Sigma–Aldrich). The injection was administered weekly throughout the duration of the experiment. Mice were anesthetized with 0.5 mL chloral hydrate per 100 g body weight and inoculated with 20 μL of 5 × 10⁸ CFU/mL C. albicans administered by mechanical pipette into the vaginal lumen, close to the cervix.

Vaginal lavage was performed on the mice using 500 μL PBS and the fungal burden in lavage fluid was assessed. Vaginal lavage obtained at different time-points after infection were treated with protease inhibitors and centrifuged at 600 × g. Supernatants were recovered and stored at -20°C, and the pellets were fixed with 4% paraformaldehyde (PFA). To assess the number of polymorphonuclear cells in vaginal wash fluid, the cells were incubated with rat anti-mouse Ly6G-Percp Cy5.5, CD4-FITC, CD8-APC, F4/80-PE for 30 min on ice. Then the cells were analyzed by flow cytometry.

Supernatants from vaginal lavage obtained from co-infected and mono-infected mice were tested for expression levels of IL-1β, IL-2, IL-6, TNF-α, IFN-γ, and IL-17. Cervical-vaginal lavage solution were collected and homogenized in 500 μl of cold PBS supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin, and 0.3 μM aprotinin) using a Dounce homogenizer. The samples were then centrifuged at 4000 rpm, and the resulting supernatant was stored at -80°C. IL-1β, IL-2, IL-6, TNF-α, and IFN-γ levels in the solution were measured with Ready-SET-GO ELISA kits (eBioscience). The level of IL-17 protein was measured using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA, United States), according to the manufacturer’s directions. All samples were measured in triplicate according to the manufacturer’s protocol. The analysis of surface molecules, intracellular IL-17 in vaginal, and lymph node cells was performed by flow cytometry.

For histological analysis, the mice were sacrificed, and the vaginal tissue were removed and immediately fixed in 4% (v/v) neutral buffered formalin for 24 h. The vaginal specimens were then dehydrated, paraffin-embedded, and 3–4 μm thick sections prepared. The sections were stained with periodic acid-Schiff stain (PAS).

Results were compared using the non-parametric Kruskal–Wallis test, non-parametric Mann–Whitney Test, or the paired t-test using Prism software (GraphPad). P-values < 0.05 were considered to be statistically significant.

Candida albicans can lead to mucosal infections in VVC and RVVC. Interestingly, we found that C. albicans strains isolated from patients with RVVC hyphal development were different on Spider plate (Supplementary Figures S1A,B). This difference may be the results of differences in the duration of infection and treatment. We named C. albicans strains isolated from patients with RVVC infection without bacterial infection as CA-RVVCS and named C. albicans strains isolated from patients of RVVC with S. agalactiae co-infection as CA-RVVCC. We further analyzed clinical strains hyphal development on Spider medium. Interestingly, we found that 82% (28/34) CA-RVVCC strains hyphal development were deficient, but 47% (16/34) CA-RVVCS strains hyphal development were deficient (Supplementary Figure S2A). To explore CA-RVVCC strains hyphal development, C. albicans cells isolated from clinical samples were cultivated on Spider plate at 37°C for 3 days to induce hyphal development. As a control, the SC5314 strains (n = 2) and CA-RVVCS strains (n = 2) were cultivated on Spider plate. We found that compared with the control strains, the colonies of CA-RVVCC strains (n = 12) exhibited fewer wrinkles suggesting that hyphal development of CA-RVVCC strains appears to be a deficient (Figure 1A). On microscopic examination, the colonies of CA-RVVCC strains also exhibited a smooth appearance, which indicated impairment of hyphal development. C. albicans on the Spider plate at 37°C induced hyphal development with upward growth in the air and downward growth invading the agar. This invasive growth of C. albicans hyphae can induce host tissues damage triggering immune defense responses. We observed the hyphal growth of CA-RVVCC strains in the agar to further assess these strains hyphal development. Next, the agar was cut longitudinally to observe the invasive growth of C. albicans and hyphal morphology by microscopy. On microscopic examination, the CA-RVVCC strains hyphal morphology showed shorter and fewer in the agar, when compared with that of SC5314 and CA-RVVCS strains, suggesting that CA-RVVCC strains hyphal invasive growth were impaired (Figure 1B). To examine whether the phenotype has requirements for growth medium, we further assessed hyphal development in YPD media. These isolates were cultured in YPD medium at 37°C for 6 h, 12 h and 24 h to observe hyphal development. At different time-points hyphal morphology was observed by the microscopy. The majority of cells of the SC5314 and CA-RVVCS strains were found to have hyphal morphology. In contrast, the majority of cells of the CA-RVVCC strains were found to have yeast-like morphology, and hyphal development was found to be severely attenuated at 6 h, 12 h and 24 h (Figure 1C). The transcription factors EFG1, NDT80, ROB1, BRG1, TEC1, and BCR1 have been shown to be involved in hyphal development of C. albicans (Nobile et al., 2012). In order to identify factors contributing to the hyphal deficient phenotypes, the transcriptional levels of EFG1, NDT80, ROB1, BRG1, TEC1, and BCR1 were assessed on RT-qPCR to identify genes that regulate hyphal development. The hyphal development transcription factors EFG1, BRG1 and BCR1 were found to be downregulated in CA-RVVCC strains when compared with SC5314 and CA-RVVCS strains (Figure 1D). These findings suggested that S. agalactiae co-infection may have attenuated the hyphal development of C. albicans in VVC.

Hyphal development is an important factor in the pathogenesis of C. albicans. We found that the Saga-Eh1 strain could inhibit hyphal development in some CA-RVVCS strains (Supplementary Figure S2B). To investigate further the interaction of S. agalactiae and C. albicans, we studied hyphal development in C. albicans after co-cultured with Saga-Eh1. The SC5314 cells were harvested from overnight cultures and re-inoculated in YPD medium. Hyphal development in C. albicans was examined in SC5314 cultivated on Spider plate at 37°C for 3 days, with or without Saga-Eh1. When the Saga-Eh1 and SC5314 were grown adjacent to one another, we found that compared with the growth of SC5314 alone, there was a striking growth suppression of C. albicans in the presence of S. agalactiae. SC5314 co-culturing with Saga-Eh1 colonies exhibited fewer wrinkles when compared with SC5314 hyphal development on Spider plate suggesting that under a condition, S. agalactiae is able to depress growth of SC5314 (Figure 2A). This result indicated that Saga-Eh1 inhibited SC5314 hyphal development via non-contact suppression. Next, we analyzed the process of morphological development from yeast to hyphae in SD+10% FBS. Saga-Eh1 overnight culture supernatant was 10 times concentrated and mixed with SD+10% FBS (1:10) to culture SC5314. At the same time, SD+10% FBS mixed with 10 times concentrated E. coli culture supernatant (1:10) was the control group to observe hyphal morphological development in SC5314. Hyphal elongation in SC5314 was found to be attenuated at 3 h after mixing with Saga-Eh1 culture supernatant when compared with normal SC5314 hyphal development in E. coli culture supernatant (Figure 2B). We further examined hyphal development in different media. C. albicans strain SC5314 was co-cultured with S. agalactiae strain Saga-Eh1 in Spider, YPD+10% FBS, DMEM+10% FBS, and SD+10% FBS medium at 37°C for 4 h to induce hyphal development, followed by microscopic examination of hyphal morphology. The results showed that length of C. albicans hyphae were short in different culture media (Supplementary Figure S3A). These findings indicated that C. albicans strain SC5314 hyphal development was attenuated upon co-culture with S. agalactiae.

Hyphal aggregation is another important characteristic of C. albicans. To evaluated C. albicans hyphal aggregation, C. albicans strain SC5314 was cultured in SD+10% FBS with and without 10 times concentrated Saga-Eh1 culture supernatant in tube at 37°C for 6 h to observe SC5314 hyphal aggregation. Surprisingly, visible massive precipitation was observed in the absence of Saga-Eh1 culture supernatant tube, while only small particular precipitates were observed in the tube with Saga-Eh1 culture supernatant. Differences in the morphology of C. albicans cultured with and without Saga-Eh1 culture supernatant were also observed during microscopic examination (Figure 2C). Furthermore, we investigated the percentage of C. albicans hyphal formation in the in vitro co-culture. C. albicans was incubated for 6 h in Spider media, with gentle shaking of samples at 37°C. Hyphal and yeast counts were performed in 200 C. albicans cells under a microscope. We found that hyphal formation with co-culture of 10 times concentrated Saga-Eh1 culture supernatant was lower than that in the absence of Saga-Eh1 culture supernatant (50.29 ± 8.58% vs. 86.80 ± 9.34%, P < 0.001), which indicated that S. agalactiae suppressed C. albicans hyphal formation in vitro (Figure 2D). Collectively, these results confirmed that S. agalactiae could suppress C. albicans hyphal development in vitro.

The transcriptional network is critical to hyphal development in C. albicans. To explore the underlying mechanism of impaired hyphal development, we assessed the mRNA expressions of transcription factors associated with hyphal development using reverse transcription quantitative polymerase chain reaction (RT-qPCR). We found that the transcription level of EFG1 was decreased significantly in C. albicans co-cultured with S. agalactiae, when compared with that in C. albicans cultured without S. agalactiae, while the mRNA levels of other transcription factors were comparable to those of the controls (Figure 3A). Hyphal wall protein (Hwp1) extends to fungal growth in the host, as shown by the presence and absence of Hwp1 on hyphae and yeast (Fan et al., 2013). Hwp1 is a transglutaminase substrate which functions as an adhesin and plays an important role in the pathogenesis of candidiasis (Sundstrom et al., 2002; Martin et al., 2011; Fan et al., 2013; Staab et al., 2013). To investigate further the underlying mechanism of the effect of S. agalactiae on hyphal development in C. albicans, the expressions of transcription factors EFG1 and hyphal development marker HWP1 were assessed on RT-qPCR. Serial RT-qPCR at various time-points showed that the mRNA expressions of EFG1 and HWP1, after hyphal induction were significantly decreased on co-culture with S. agalactiae (Figure 3B). Moreover, the expression of Hwp1 in the co-culture group determined on Western blotting analysis at 60 min, 90 min and 120 min was lower than that in the control group (Figure 3C). Together, these findings suggest that co-culture with S. agalactiae attenuates hyphal development of C. albicans by downregulating EFG1-Hwp1.

Hyphal formation and adherence are pivotal for biofilm formation by C. albicans, which is involved in the pathogenesis of many infections (Andes et al., 2004; Fox and Nobile, 2012). Different components of the immune system identify hyphae by different mechanisms, which are pivotal for inducing host defense response (Gow, 2013). To explored the role of S. agalactiae in the pathogenesis of C. albicans infection, a mouse model of VVC was established. Vaginal lumen of mice were inoculated with SC5314 and Saga-Eh1 (MOI = 10). Five days post-inoculation, 500 μL PBS was used to perform mice vaginal lavage. This lavage fluid was plated on YPD and cultured to assess mice vaginal fungal burden by CFU counting. The vaginal fungal burden in mice infected with C. albicans and Saga-Eh1 was greater than that in mice without Saga-Eh1 (5.75 × 10⁴ ± 420 per mouse vs. 2.9 × 10⁴ ± 191 per mouse, P < 0.05) (Figure 4A). Neutrophils are an important immune cell and were detected to evaluated host immune defense reaction. In VVC mouse vaginal lumen, neutrophil cells counts in vaginal lavage fluid showed significant difference between-group differences (1234 ± 122.5 per mouse vs. 769 ± 130.3 per mouse, P < 0.05) (Figure 4B). Furthermore, histopathological examination of vaginal mucosa stained with PAS (Periodic acid–Schiff staining, PAS) on day 5 post-inoculation showed massive C. albicans colonization in VVC mice co-infected with Saga-Eh1, when compared with mice without Saga-Eh1 co-infection (Figure 4C). To confirm C. albicans colonization on vaginal mucosa, SC5314-GFP strain was used to infect mice to establish a mouse model of VVC. On fluorescence microscopy, more number of colonies of SC5314-GFP were observed on vaginal mucosa of mice co-infected with Saga-Eh1 as compared to that in vaginal mucosa of the control mice (Figure 4D). Analysis of the total photon emission showed a significant reduction in the fungal load on vaginal mucosa of mice co-infected with Saga-Eh1 as compared to that in vaginal mucosa of control mice on day 5 post-inoculation. Similar results were observed on PAS staining in the same mice.

Colonization of the vaginal mucosal by C. albicans is known to depend on adhesion of C. albicans and on the host-immune clearance (Gow et al., 2012). The adherence of fungal cells to host epithelial cells during the initial contact is important because it determines the subsequent fungal-host interactions.

The adhesion of the fungus to epithelial cells is a complex process mediated by hyphal-associated factors, such as Hwp1 and Als3 (Martin et al., 2011; Mech et al., 2014; Moyes et al., 2015). Human epithelial colorectal adenocarcinoma cells (Coca2 cells) and human epidermoid carcinoma cells (A431 cells) were cultured in Dulbecco’s Modified Eagle medium (DMEM)+10% FBS medium at 37°C; then C. albicans strain SC5314 was cultured with or without Saga-Eh1 in transwell at 37°C. After 2 h of co-culture, no significant difference was observed in the counts of adherent cells between the two groups. This indicated that S. agalactiae did not affect adhesive ability of C. albicans (Supplementary Figure S3B).

Neutrophils and TH17 cells play an important role in the immune response against colonization of vaginal mucosal surface by C. albicans (Pietrella et al., 2011; Hopke et al., 2016; Salvatori et al., 2016). To explore host immune responses induced by C. albicans and S. agalactiae co-infection, Saga-Eh1and SC5314 were inoculated into the vaginal mucosa of mice and the immune responses were monitored using flow cytometry. The results showed presence of different types of cells in the vaginal fluid of the infected mice, especially neutrophils and epithelial cells. On day 5 post-inoculation, vaginal mucosal immune cells were detected by flow cytometry. The results showed that the percentage of neutrophils cells increased from 65.53 ± 14.45% to 71.67 ± 15.92% (P > 0.05), and the percentage of macrophages decreased from 39.53 ± 16.37% to 35.80 ± 13.93% (P > 0.05); however, these percentages were not significantly different between the co-infected mice and controls (Figure 5A). During the early phase of vaginal mucosal infection, neutrophils cells were recruited and extravasated using pro-inflammatory mediators, which was followed by activation of TH17 response (Kolaczkowska and Kubes, 2013). TH17 cells belong to a lineage that is different from the Th1 and Th2 cells, and these are characterized by the production of IL-17. We observed that the number of TH17 T lymphocytes were significantly decreased in mice co-infected with Saga-Eh1 as compared to that in control mice (89.28 ± 3.38% vs. 69.93 ± 5.71%, P < 0.05) (Figure 5B). However, no significant differences were observed with respect to CD4+ T cells (20.83 ± 7.66% vs. 22.6 ± 6.96%, P > 0.05), and CD8+ T cells (5.62 ± 4.43% vs. 3.395 ± 1.94%, P > 0.05) (data not shown).

The IL-17 secreted by activated TH17 T cells has been shown to play a critical role in protecting against mucosal infections, and particularly against mucosal candidiasis (Pietrella et al., 2011; Puel et al., 2011). The IL-17 response correlated with the hyphal formation ability of C. albicans, which stimulates macrophages, neutrophils, and other innate immune cells, hence priming the host-innate immune response and inducing cytokine production for priming the TH17 response. We further investigated the related inflammatory cytokines in a mouse model of vaginal infection. Production of cytokines was higher in the cervical-vaginal lavage solution of mice co-infected with Saga-Eh1 compared with those given mono-infection with SC5314 as IL-1β (114.65 ± 2.66 pg/mL vs. 122.6 ± 6.96 pg/mL, P < 0.05); IL-6 (84.60 ± 1.05 pg/mL vs. 86.01 ± 2.21 pg/mL, P > 0.05); and TNF-α (78.83 ± 3.43 pg/mL vs. 81.6 ± 4.96 pg/mL, P > 0.05)] (Figure 5C). Activated TH17 cells produced more IL-17 to enable C. albicans clearance. However, post-challenge there was decreased production of IL-17 (42.10 ± 1.43 pg/mL vs. 31.6 ± 4.96 pg/mL, P < 0.05) in the vaginal washes from co-infected Saga-Eh1 mice as compared to that in mice with mono-infection SC5314 (Figure 5C). Collectively, these results demonstrated that TH17 T cells immune response in vaginal mucosa was decreased due to S. agalactiae co-infection.